The adhesion of cells to extracellular matrix molecules plays a critical role in many important biological processes including embryogenesis, cell-mediated immunity, wound healing, and malignant transformation. Our long term goal is to understand how a cell couples events at the adhesive membrane to intracellular signaling pathways that regulate such diverse processes as cell locomotion and gene expression. One protein that has emerged as an excellent candidate for participating in information transfer at sites of cell-substratum adhesion is zyxin. Zyxin is a low abundance LIM domain protein that is co-localized with extracelluar matrix receptors; zyxin displays a number of properties consistent with a role as an intracellular signal transducer. Experiments performed in the current funding period have led us to postulate that zyxin is involved in defining active zones of actin assembly and in mediating communication between the nucleus and the adhesive membrane. The role of zyxin in cell migration and spreading, cellular processes that depend on spatially restricted actin assembly and organization, will be examined by chromophore-assisted laser inactivation. Site- directed mutagenesis of zyxin and in vivo competition experiments with peptides that block zyxin's ability to associate with specific binding partners will be employed to map domains that are essential for zyxin s ability to stimulate actin assembly and reorganization. In addition, the role of zyxin in cytoskeletal rearrangements involving Rho-GTPases will be assessed. Zyxin displays a nuclear export signal and shuttles between the nucleus and the adhesive membrane. Mapping studies will be performed to define the mechanisms by which zyxin is targeted to the nucleus. Proteins that are complexed with zyxin in the nucleus will be identified and characterized. These studies will provide insight into the molecular mechanisms and physiological significance of zyxin's ability to transit between sites of cell adhesion and the nucleus. Finally, zyxin function will be eliminated by targeted gene disruption in mouse embryonic stem cells; the consequences of loss of zyxin expression for cell adhesion, cell morphology, cell migration, and cell differentiation will be defined. The combination of biochemical, cellular, and molecular genetic approaches proposed in this application will provide insight into the role of zyxin in adhesion-stimulated changes in cell behavior.